RESUMEN
Non-small cell lung cancers (NSCLCs) demonstrate intrinsic resistance to cell death, even after chemotherapy. Previous work suggested defective nuclear translocation of active caspase-3 in observed resistance to cell death. We have identified mitogen-activated protein kinase-activated protein kinase 2 (MK2; encoded by the gene MAPKAPK2) is required for caspase-3 nuclear translocation in the execution of apoptosis in endothelial cells. The objective was to determine MK2 expression in NSCLCs and the association between MK2 and clinical outcomes in patients with NSCLC. Clinical and MK2 mRNA data were extracted from two demographically distinct NSCLC clinical cohorts, North American (The Cancer Genome Atlas, TCGA) and East Asian (EA). Tumor responses following first round of chemotherapy were dichotomized as clinical response (complete response, partial response, and stable disease) or progression of disease. Multivariable survival analyses were performed using Cox proportional hazard ratios and Kaplan-Meier curves. NSCLC exhibited lower MK2 expression than SCLC cell lines. In patients, lower tumor MK2 transcript levels were observed in those presenting with late-stage NSCLC. Higher MK2 expression was associated with clinical response following initial chemotherapy and independently associated with improved 2-yr survival in two distinct cohorts, 0.52 (0.28-0.98) and 0.1 (0.01-0.81), TCGA and EA, respectively, even after adjusting for common oncogenic driver mutations. Survival benefit of higher MK2 expression was unique to lung adenocarcinoma when comparing across various cancers. This study implicates MK2 in apoptosis resistance in NSCLC and suggests prognostic value of MK2 transcript levels in patients with lung adenocarcinoma.
Asunto(s)
Adenocarcinoma del Pulmón , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Caspasa 3/uso terapéutico , Células Endoteliales , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genéticaRESUMEN
We have previously identified mitogen-activated protein kinase-activated protein kinase 2 (MK2) is required for caspase-3 nuclear translocation in the execution of apoptosis; however, little is known of the underlying mechanisms. Therefore, we sought to determine the role of kinase and nonkinase functions of MK2 in promoting nuclear translocation of caspase-3. We identified two non-small cell lung cancer cell lines for use in these experiments based on low MK2 expression. Wild-type, enzymatic and cellular localization mutant MK2 constructs were expressed using adenoviral infection. Cell death was evaluated by flow cytometry. In addition, cell lysates were harvested for protein analyses. Phosphorylation of caspase-3 was determined using two-dimensional gel electrophoresis followed by immunoblotting and in vitro kinase assay. Association between MK2 and caspase-3 was evaluated using proximity-based biotin ligation assays and co-immunoprecipitation. Overexpression of MK2 resulted in nuclear translocation of caspase-3 and caspase-3-mediated apoptosis. MK2 directly phosphorylates caspase-3; however, phosphorylation status of caspase-3 or MK2-dependent phosphorylation of caspase-3 did not alter caspase-3 activity. The enzymatic function of MK2 was dispensable in nuclear translocation of caspase-3. MK2 and caspase-3 associated together and a nonenzymatic function of MK2, chaperoned nuclear trafficking, is required for caspase-3-mediated apoptosis. Taken together, our results demonstrate a nonenzymatic role for MK2 in the nuclear translocation of caspase-3. Furthermore, MK2 may function as a molecular switch in regulating the transition between the cytosolic and nuclear functions of caspase-3.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Apoptosis , Caspasa 3/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Coronaviruses (CoVs) assemble by budding into the lumen of the early Golgi complex prior to exocytosis. The small CoV envelope (E) protein plays roles in assembly, virion release, and pathogenesis. CoV E has a single hydrophobic domain (HD), is targeted to Golgi membranes, and has cation channel activity in vitro The E protein from avian infectious bronchitis virus (IBV) has dramatic effects on the secretory system, which require residues in the HD. Mutation of the HD of IBV E in a recombinant virus background results in impaired growth kinetics, impaired release of infectious virions, accumulation of IBV spike (S) protein on the plasma membrane compared to wild-type (WT) IBV-infected cells, and aberrant cleavage of IBV S on virions. We previously reported the formation of two distinct oligomeric pools of IBV E in transfected and infected cells. Disruption of the secretory pathway by IBV E correlates with a form that is likely monomeric, suggesting that the effects on the secretory pathway are independent of E ion channel activity. Here, we present evidence suggesting that the monomeric form of IBV E correlates with an increased Golgi luminal pH. Infection with IBV or expression of IBV E induces neutralization of Golgi pH, promoting a model in which IBV E alters the secretory pathway through interaction with host cell factors, protecting IBV S from premature cleavage and leading to the efficient release of infectious virus from the cells. This is the first demonstration of a coronavirus-induced alteration in the microenvironment of the secretory pathway.IMPORTANCE Coronaviruses are important human pathogens with significant zoonotic potential. Progress has been made toward identifying potential vaccine candidates for highly pathogenic human CoVs, including the use of attenuated viruses that lack the CoV E protein or express E mutants. However, no approved vaccines or antiviral therapeutics exist. Understanding the role of the CoV E protein in virus assembly and release is thus an important prerequisite for potential vaccines as well as in identifying novel antiviral therapeutics.
Asunto(s)
Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Animales , Bronquitis/inmunología , Bronquitis/virología , Membrana Celular/metabolismo , Chlorocebus aethiops , Coronavirus/patogenicidad , Proteínas de la Envoltura de Coronavirus , Infecciones por Coronavirus/virología , Aparato de Golgi/fisiología , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Virus de la Bronquitis Infecciosa/inmunología , Vías Secretoras , Células Vero , Proteínas del Envoltorio Viral/fisiología , Virión/metabolismo , Ensamble de Virus , Virosis/metabolismoRESUMEN
Noncanonical roles for caspase-3 are emerging in the fields of cancer and developmental biology. However, little is known of nonapoptotic functions of caspase-3 in most cell types. We have recently demonstrated a disassociation between caspase-3 activation and execution of apoptosis with accompanying cytoplasmic caspase-3 sequestration and preserved endothelial barrier function. Therefore, we tested the hypothesis that nonapoptotic caspase-3 activation promotes endothelial barrier integrity. Human lung microvascular endothelial cells were exposed to thrombin, a nonapoptotic stimulus, and endothelial barrier function was assessed using electric cell-substrate impedance sensing. Actin cytoskeletal rearrangement and paracellular gap formation were assessed using phalloidin staining. Cell stiffness was evaluated using magnetic twisting cytometry. In addition, cell lysates were harvested for protein analyses. Caspase-3 was inhibited pharmacologically with pan-caspase and a caspase-3-specific inhibitor. Molecular inhibition of caspase-3 was achieved using RNA interference. Cells exposed to thrombin exhibited a cytoplasmic activation of caspase-3 with transient and nonapoptotic decrease in endothelial barrier function as measured by a drop in electrical resistance followed by a rapid recovery. Inhibition of caspases led to a more pronounced and rapid drop in thrombin-induced endothelial barrier function, accompanied by increased endothelial cell stiffness and paracellular gaps. Caspase-3-specific inhibition and caspase-3 knockdown both resulted in more pronounced thrombin-induced endothelial barrier disruption. Taken together, our results suggest cytoplasmic caspase-3 has nonapoptotic functions in human endothelium and can promote endothelial barrier integrity.
Asunto(s)
Caspasa 3/metabolismo , Células Endoteliales/citología , Endotelio Vascular/metabolismo , Mucosa Respiratoria/citología , Uniones Estrechas/efectos de los fármacos , Citoesqueleto de Actina/fisiología , Permeabilidad Capilar/efectos de los fármacos , Caspasa 3/genética , Células Cultivadas , Impedancia Eléctrica , Endotelio Vascular/citología , Humanos , Pulmón/citología , Interferencia de ARN , ARN Interferente Pequeño/genética , Trombina/farmacologíaRESUMEN
Enveloped viruses gain entry into host cells by fusing with cellular membranes, a step that is required for virus replication. Coronaviruses, including the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and infectious bronchitis virus (IBV), fuse at the plasma membrane or use receptor-mediated endocytosis and fuse with endosomes, depending on the cell or tissue type. The virus spike (S) protein mediates fusion with the host cell membrane. We have shown previously that an Abelson (Abl) kinase inhibitor, imatinib, significantly reduces SARS-CoV and MERS-CoV viral titres and prevents endosomal entry by HIV SARS S and MERS S pseudotyped virions. SARS-CoV and MERS-CoV are classified as BSL-3 viruses, which makes experimentation into the cellular mechanisms involved in infection more challenging. Here, we use IBV, a BSL-2 virus, as a model for studying the role of Abl kinase activity during coronavirus infection. We found that imatinib and two specific Abl kinase inhibitors, GNF2 and GNF5, reduce IBV titres by blocking the first round of virus infection. Additionally, all three drugs prevented IBV S-induced syncytia formation prior to the hemifusion step. Our results indicate that membrane fusion (both virus-cell and cell-cell) is blocked in the presence of Abl kinase inhibitors. Studying the effects of Abl kinase inhibitors on IBV will be useful in identifying the host cell pathways required for coronavirus infection. This will provide an insight into possible therapeutic targets to treat infections by current as well as newly emerging coronaviruses.
Asunto(s)
Endosomas/virología , Virus de la Bronquitis Infecciosa/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del Virus , Animales , Antivirales/farmacología , Benzamidas/farmacología , Membrana Celular , Chlorocebus aethiops , Mesilato de Imatinib/farmacología , Virus de la Bronquitis Infecciosa/metabolismo , Pirimidinas/farmacología , Glicoproteína de la Espiga del Coronavirus/genética , Células Vero , Replicación ViralRESUMEN
Sterol regulatory element-binding protein (SREBP) transcription factors are central regulators of cellular lipid homeostasis and activate expression of genes required for fatty acid, triglyceride, and cholesterol synthesis and uptake. SREBP cleavage activating protein (SCAP) plays an essential role in SREBP activation by mediating endoplasmic reticulum (ER)-to-Golgi transport of SREBP. In the Golgi, membrane-bound SREBPs are cleaved sequentially by the site-1 and site-2 proteases. Recent studies have shown a requirement for the SREBP pathway in the development of fatty liver disease and tumor growth, making SCAP a target for drug development. Fatostatin is a chemical inhibitor of the SREBP pathway that directly binds SCAP and blocks its ER-to-Golgi transport. In this study, we determined that fatostatin blocks ER exit of SCAP and showed that inhibition is independent of insulin-induced gene proteins, which function to retain the SCAP-SREBP complex in the ER. Fatostatin potently inhibited cell growth, but unexpectedly exogenous lipids failed to rescue proliferation of fatostatin-treated cells. Furthermore, fatostatin inhibited growth of cells lacking SCAP Using a vesicular stomatitis virus glycoprotein (VSVG) trafficking assay, we demonstrated that fatostatin delays ER-to-Golgi transport of VSVG. In summary, fatostatin inhibited SREBP activation, but fatostatin additionally inhibited cell proliferation through both lipid-independent and SCAP-independent mechanisms, possibly by general inhibition of ER-to-Golgi transport.
Asunto(s)
Retículo Endoplásmico/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Piridinas/farmacología , Tiazoles/farmacología , Animales , Células CHO , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cricetinae , Cricetulus , Evaluación Preclínica de Medicamentos , Retículo Endoplásmico/efectos de los fármacos , Aparato de Golgi/metabolismo , Células HEK293 , Humanos , Transporte de Proteínas/efectos de los fármacosRESUMEN
UNLABELLED: Coronaviruses (CoVs) assemble by budding into the lumen of the early Golgi complex prior to exocytosis. The small CoV envelope (E) protein plays roles in assembly, virion release, and pathogenesis. CoV E has a single hydrophobic domain (HD), is targeted to Golgi complex membranes, and has cation channel activity in vitro. However, the precise functions of the CoV E protein during infection are still enigmatic. Structural data for the severe acute respiratory syndrome (SARS)-CoV E protein suggest that it assembles into a homopentamer. Specific residues in the HD regulate the ion-conducting pore formed by SARS-CoV E in artificial bilayers and the pathogenicity of the virus during infection. The E protein from the avian infectious bronchitis virus (IBV) has dramatic effects on the secretory system which require residues in the HD. Here, we use the known structural data from SARS-CoV E to infer the residues important for ion channel activity and the oligomerization of IBV E. We present biochemical data for the formation of two distinct oligomeric pools of IBV E in transfected and infected cells and the residues required for their formation. A high-order oligomer of IBV E is required for the production of virus-like particles (VLPs), implicating this form of the protein in virion assembly. Additionally, disruption of the secretory pathway by IBV E correlates with a form that is likely monomeric, suggesting that the effects on the secretory pathway are independent of E ion channel activity. IMPORTANCE: CoVs are important human pathogens with significant zoonotic potential, as demonstrated by the emergence of SARS-CoV and Middle East respiratory syndrome (MERS)-CoV. Progress has been made toward identifying potential vaccine candidates in mouse models of CoV infection, including the use of attenuated viruses that lack the CoV E protein or express E-protein mutants. However, no approved vaccines or antiviral therapeutics exist. We previously reported that the hydrophobic domain of the IBV E protein, a putative viroporin, causes disruption of the mammalian secretory pathway when exogenously expressed in cells. Understanding the mechanism of this disruption could lead to the identification of novel antiviral therapeutics. Here, we present biochemical evidence for two distinct oligomeric forms of IBV E, one essential for assembly and the other with a role in disruption of the secretory pathway. Discovery of two forms of CoV E protein will provide additional targets for antiviral therapeutics.
Asunto(s)
Multimerización de Proteína/fisiología , Síndrome Respiratorio Agudo Grave/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/fisiología , Proteínas del Envoltorio Viral/metabolismo , Ensamble de Virus/fisiología , Animales , Chlorocebus aethiops , Células HeLa , Humanos , Ratones , Estructura Cuaternaria de Proteína , Síndrome Respiratorio Agudo Grave/genética , Células Vero , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas ViroporinasRESUMEN
Apoptosis is a key pathologic feature in acute lung injury. Animal studies have demonstrated that pathways regulating apoptosis are necessary in the development of acute lung injury, and that activation of p38 mitogen-activated protein kinase (MAPK) is linked to the initiation of the apoptotic cascade. In this study, we assessed the role of the MAPK-activated protein kinase (MK) 2, one of p38 MAPK's immediate downstream effectors, in the development of apoptosis in an animal model of LPS-induced pulmonary vascular permeability. Our results indicate that wild-type (WT) mice exposed to LPS demonstrate increased apoptosis, as evidenced by cleavage of caspase 3 and poly (ADP-ribose) polymerase 1 and increased deoxynucleotidyl transferase-mediated dUDP nick-end labeling staining, which is accompanied by increases in markers of vascular permeability. In contrast, MK2(-/-) mice are protected from pulmonary vascular permeability and apoptosis in response to LPS. Although there was no difference in activation of caspase 3 in MK2(-/-) compared with WT mice, interestingly, cleaved caspase 3 translocated to the nucleus in WT mice while it remained in the cytosol of MK2(-/-) mice in response to LPS. In separate experiments, LPS-induced apoptosis in human lung microvascular endothelial cells was also associated with nuclear translocation of cleaved caspase 3 and apoptosis, which were both prevented by MK2 silencing. In conclusion, our data suggest that MK2 plays a critical role in the development of apoptosis and pulmonary vascular permeability, and its effects on apoptosis are in part related to its ability to regulate nuclear translocation of cleaved caspase 3.
Asunto(s)
Apoptosis/fisiología , Caspasa 3/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Pulmón/irrigación sanguínea , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Permeabilidad Capilar , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Poli(ADP-Ribosa) PolimerasasRESUMEN
The coronavirus E protein is a small membrane protein with a single predicted hydrophobic domain (HD), and has a poorly defined role in infection. The E protein is thought to promote virion assembly, which occurs in the Golgi region of infected cells. It has also been implicated in the release of infectious particles after budding. The E protein has ion channel activity in vitro, although a role for channel activity in infection has not been established. Furthermore, the membrane topology of the E protein is of considerable debate, and the protein may adopt more than one topology during infection. We previously showed that the HD of the infectious bronchitis virus (IBV) E protein is required for the efficient release of infectious virus, an activity that correlated with disruption of the secretory pathway. Here we report that a single residue within the hydrophobic domain, Thr16, is required for secretory pathway disruption. Substitutions of other residues for Thr16 were not tolerated. Mutations of Thr16 did not impact virus assembly as judged by virus-like particle production, suggesting that alteration of secretory pathway and assembly are independent activities. We also examined how the membrane topology of IBV E affected its function by generating mutant versions that adopted either a transmembrane or membrane hairpin topology. We found that a transmembrane topology was required for disrupting the secretory pathway, but was less efficient for virus-like particle production. The hairpin version of E was unable to disrupt the secretory pathway or produce particles. The findings reported here identify properties of the E protein that are important for its function, and provide insight into how the E protein may perform multiple roles during infection.
Asunto(s)
Infecciones por Coronavirus/virología , Virus de la Bronquitis Infecciosa/química , Virus de la Bronquitis Infecciosa/metabolismo , Vías Secretoras , Proteínas del Envoltorio Viral/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , Línea Celular Tumoral , Infecciones por Coronavirus/metabolismo , Aparato de Golgi/ultraestructura , Células HeLa , Humanos , Virus de la Bronquitis Infecciosa/genética , Virus de la Bronquitis Infecciosa/patogenicidad , Mutación , Estructura Terciaria de Proteína , Transporte de Proteínas , Alineación de Secuencia , Proteínas del Envoltorio Viral/química , Ensamble de VirusRESUMEN
Golgin-160 is a member of the golgin family of proteins, which have been implicated in the maintenance of Golgi structure and in vesicle tethering. Golgin-160 is atypical; it promotes post-Golgi trafficking of specific cargo proteins, including the ß-1 adrenergic receptor (ß1AR), a G protein-coupled receptor. Here we show that golgin-160 binds directly to the third intracellular loop of ß1AR and that this binding depends on three basic residues in this loop. Mutation of the basic residues does not affect trafficking of ß1AR from the endoplasmic reticulum through the Golgi complex, but results in reduced steady-state levels at the plasma membrane. We hypothesize that golgin-160 promotes incorporation of ß1AR into specific transport carriers at the trans-Golgi network to ensure efficient delivery to the cell surface. These results add to our understanding of the biogenesis of ß1AR, and suggest a novel point of regulation for its delivery to the plasma membrane.
Asunto(s)
Autoantígenos/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Secuencia de Aminoácidos , Autoantígenos/química , Autoantígenos/genética , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Matriz de Golgi , Células HeLa , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Receptores Adrenérgicos beta 1/químicaRESUMEN
In mammalian cells, the Golgi complex has an elaborate structure consisting of stacked, flattened cisternal membranes collected into a ribbon in the center of the cell. Amazingly, the flattened cisternae can rapidly dilate to accommodate large cargo as it traffics through the organelle. The mechanism by which this occurs is unknown. Exocytosis of large cargo is essential for many physiological processes, including collagen and lipoprotein secretion, and defects in the process lead to disease. In addition, enveloped viruses that bud into the endoplasmic reticulum or Golgi complex must also be transported through Golgi cisternae for secretion from the infected cell. This review summarizes our understanding of intra-Golgi transport of large cargo, and outlines current questions open for experimentation.
Asunto(s)
Aparato de Golgi/química , Aparato de Golgi/metabolismo , Animales , Transporte Biológico , Retículo Endoplásmico/metabolismo , HumanosRESUMEN
The mammalian Golgi apparatus is composed of multiple stacks of cisternal membranes organized laterally into a polarized ribbon. Furthermore, trans-Golgi membranes come in close apposition with ER (endoplasmic reticulum) membranes to form ER-trans-Golgi contact sites, which may facilitate transfer of newly synthesized ceramide from the ER to SM (sphingomyelin) synthase at the trans-Golgi via CERT (ceramide transfer protein). CERT interacts with both ER and Golgi membranes, and together with Golgi morphology contributes to efficient SM synthesis. In the present study, we show that Golgi disassembly during pro-apoptotic stress induced by TNFα (tumour necrosis factor α) and anisomycin results in decreased levels of CERT at the Golgi region. This is accompanied by a caspase-dependent loss of full-length CERT and reduction in de novo SM synthesis. In vitro, CERT is cleaved by caspases 2, 3 and 9. Truncated versions of CERT corresponding to fragments generated by caspase 2 cleavage at Asp213 were mislocalized and did not promote efficient de novo SM synthesis. Thus it is likely that during cellular stress, disassembly of Golgi structure together with inactivation of CERT by caspases causes a reduction in ceramide trafficking and SM synthesis, and could contribute to the cellular response to pro-apoptotic stress.
Asunto(s)
Apoptosis/fisiología , Caspasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Sustitución de Aminoácidos , Ácido Aspártico/química , Sitios de Unión , Caspasa 2/metabolismo , Ceramidas/metabolismo , Cisteína Endopeptidasas/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Mutagénesis Sitio-Dirigida , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Esfingomielinas/metabolismo , Estrés Fisiológico , Especificidad por SustratoRESUMEN
S-Palmitoylation of G protein-coupled receptors (GPCRs) is a prevalent modification, contributing to the regulation of receptor function. Despite its importance, the palmitoylation status of the ß(1)-adrenergic receptor, a GPCR critical for heart function, has never been determined. We report here that the ß(1)-adrenergic receptor is palmitoylated on three cysteine residues at two sites in the C-terminal tail. One site (proximal) is adjacent to the seventh transmembrane domain and is a consensus site for GPCRs, and the other (distal) is downstream. These sites are modified in different cellular compartments, and the distal palmitoylation site contributes to efficient internalization of the receptor following agonist stimulation. Using a bioorthogonal palmitate reporter to quantify palmitoylation accurately, we found that the rates of palmitate turnover at each site are dramatically different. Although palmitoylation at the proximal site is remarkably stable, palmitoylation at the distal site is rapidly turned over. This is the first report documenting differential dynamics of palmitoylation sites in a GPCR. Our results have important implications for function and regulation of the clinically important ß(1)-adrenergic receptor.
Asunto(s)
Cisteína/metabolismo , Lipoilación/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Receptores Adrenérgicos beta 1/metabolismo , Cisteína/genética , Células HEK293 , Humanos , Estructura Terciaria de Proteína , Receptores Adrenérgicos beta 1/genéticaRESUMEN
The coronavirus (CoV) E protein plays an important role in virus assembly. The E protein is made in excess during infection and has been shown to have ion channel activity in planar lipid bilayers. However, a role in infection for the unincorporated E or its ion channel activity has not been described. To further investigate the function of the infectious bronchitis virus (IBV) E protein, we developed a recombinant version of IBV in which the E protein was replaced by a mutant containing a heterologous hydrophobic domain. The mutant virus, IBV-EG3, was defective in release of infectious virus particles. Further characterization of IBV-EG3 revealed that damaged particles appeared to accumulate intracellularly. The phenotype of IBV-EG3 suggested that the hydrophobic domain of IBV E may be important for the forward trafficking of cargo, so we determined whether IBV E facilitated the delivery of cargo to the plasma membrane. Surprisingly, we found that IBV E, but not EG3, dramatically reduced the delivery of cargo to the plasma membrane by impeding movement through the Golgi complex. Furthermore, we observed that overexpression of IBV E, but not EG3, induced the disassembly of the Golgi complex. Finally, we determined that the delivery of IBV S to the plasma membrane was reduced in cells infected with wild-type-IBV compared to those infected with IBV-EG3. Our results indicated that the hydrophobic domain of IBV E alters the host secretory pathway to the apparent advantage of the virus.
Asunto(s)
Virus de la Bronquitis Infecciosa/fisiología , Vías Secretoras , Proteínas del Envoltorio Viral/fisiología , Liberación del Virus , Animales , Membrana Celular/química , Membrana Celular/metabolismo , Chlorocebus aethiops , Aparato de Golgi/química , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Virus de la Bronquitis Infecciosa/genética , Estructura Terciaria de Proteína , Recombinación Genética , Células Vero , Proteínas del Envoltorio Viral/genéticaRESUMEN
The 2003 global outbreak of progressive respiratory failure was caused by a newly emerged virus, severe acute respiratory syndrome coronavirus (SARS-CoV). In contrast to many well-studied enveloped viruses that assemble and bud at the plasma membrane, coronaviruses assemble by budding into the lumen of the endoplasmic reticulum-Golgi intermediate compartment and are released from the cell by exocytosis. For this to occur, the viral envelope proteins must be efficiently targeted to the Golgi region of the secretory pathway. Although the envelope protein (E) makes up only a small percentage of the viral envelope, it plays an important, as-yet-undefined role in virus production. To dissect the targeting of the SARS-CoV E protein to the Golgi region, we exogenously expressed the protein and various mutants from cDNA and determined their localization using immunofluorescence microscopy and biochemical assays. We show that the cytoplasmic tail of the SARS-CoV E protein is sufficient to redirect a plasma membrane protein to the Golgi region. Through site-directed mutagenesis, we demonstrate that a predicted beta-hairpin structural motif in the tail is sufficient for Golgi complex localization of a reporter protein. This motif is conserved in E proteins of beta and gamma coronaviruses (formerly referred to as group 2 and 3 coronaviruses), where it also functions as a Golgi complex-targeting signal. Dissecting the mechanism of targeting of the SARS-CoV E protein will lead to a better understanding of its role in the assembly and release of virions.
Asunto(s)
Aparato de Golgi/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/metabolismo , Transducción de Señal , Proteínas del Envoltorio Viral/química , Secuencias de Aminoácidos , Células HeLa , Humanos , Microscopía Fluorescente , Mutación , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Transfección , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Proteínas ViroporinasRESUMEN
RATIONALE: Gap junctions mediate cell-to-cell electric coupling of cardiomyocytes. The primary gap junction protein in the working myocardium, connexin43 (Cx43), exhibits increased localization at the lateral membranes of cardiomyocytes in a variety of heart diseases, although the precise location and function of this population is unknown. OBJECTIVE: To define the subcellular location of lateralized gap junctions at the light and electron microscopic level, and further characterize the biochemical regulation of gap junction turnover. METHODS AND RESULTS: By electron microscopy, we characterized gap junctions formed between cardiomyocyte lateral membranes in failing canine ventricular myocardium. These gap junctions were varied in structure and appeared to be extensively internalizing. Internalized gap junctions were incorporated into multilamellar membrane structures, with features characteristic of autophagosomes. Intracellular Cx43 extensively colocalized with the autophagosome marker GFP-LC3 when both proteins were exogenously expressed in HeLa cells, and endogenous Cx43 colocalized with GFP-LC3 in neonatal rat ventricular myocytes. Furthermore, a distinct phosphorylated form of Cx43, as well as the autophagosome-targeted form of LC3 (microtubule-associated protein light chain 3) targeted to lipid rafts in cardiac tissue, and both were increased in heart failure. CONCLUSIONS: Our data demonstrate a previously unrecognized pathway of gap junction internalization and degradation in the heart and identify a cellular pathway with potential therapeutic implications.
Asunto(s)
Conexina 43/metabolismo , Uniones Comunicantes/metabolismo , Uniones Comunicantes/ultraestructura , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Miocardio/metabolismo , Miocardio/ultraestructura , Animales , Autofagia , Conexina 43/genética , Modelos Animales de Enfermedad , Perros , Células HeLa , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/ultraestructura , Humanos , Microdominios de Membrana/metabolismo , Microdominios de Membrana/ultraestructura , Microscopía Confocal , Microscopía Electrónica de Transmisión , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Fosforilación , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
BackgroundSome clinical features of severe COVID-19 represent blood vessel damage induced by activation of host immune responses initiated by the coronavirus SARS-CoV-2. We hypothesized autoantibodies against angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 receptor expressed on vascular endothelium, are generated during COVID-19 and are of mechanistic importance.MethodsIn an opportunity sample of 118 COVID-19 inpatients, autoantibodies recognizing ACE2 were detected by ELISA. Binding properties of anti-ACE2 IgM were analyzed via biolayer interferometry. Effects of anti-ACE2 IgM on complement activation and endothelial function were demonstrated in a tissue-engineered pulmonary microvessel model.ResultsAnti-ACE2 IgM (not IgG) autoantibodies were associated with severe COVID-19 and found in 18/66 (27.2%) patients with severe disease compared with 2/52 (3.8%) of patients with moderate disease (OR 9.38, 95% CI 2.38-42.0; P = 0.0009). Anti-ACE2 IgM autoantibodies were rare (2/50) in non-COVID-19 ventilated patients with acute respiratory distress syndrome. Unexpectedly, ACE2-reactive IgM autoantibodies in COVID-19 did not undergo class-switching to IgG and had apparent KD values of 5.6-21.7 nM, indicating they are T cell independent. Anti-ACE2 IgMs activated complement and initiated complement-binding and functional changes in endothelial cells in microvessels, suggesting they contribute to the angiocentric pathology of COVID-19.ConclusionWe identify anti-ACE2 IgM as a mechanism-based biomarker strongly associated with severe clinical outcomes in SARS-CoV-2 infection, which has therapeutic implications.FUNDINGBill & Melinda Gates Foundation, Gates Philanthropy Partners, Donald B. and Dorothy L. Stabler Foundation, and Jerome L. Greene Foundation; NIH R01 AR073208, R01 AR069569, Institutional Research and Academic Career Development Award (5K12GM123914-03), National Heart, Lung, and Blood Institute R21HL145216, and Division of Intramural Research, National Institute of Allergy and Infectious Diseases; National Science Foundation Graduate Research Fellowship (DGE1746891).
Asunto(s)
Enzima Convertidora de Angiotensina 2 , COVID-19 , Autoanticuerpos , Células Endoteliales , Humanos , Inmunoglobulina M , SARS-CoV-2RESUMEN
Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes 3 major envelope proteins: spike (S), membrane (M), and envelope (E). Previous work identified a dibasic endoplasmic reticulum retrieval signal in the cytoplasmic tail of SARS-CoV S that promotes efficient interaction with SARS-CoV M. The dibasic signal was shown to be important for concentrating S near the virus assembly site rather than for direct interaction with M. Here, we investigated the sequence requirements of the SARS-CoV M protein that are necessary for interaction with SARS-CoV S. The SARS-CoV M tail was shown to be necessary for S localization in the Golgi region when the proteins were exogenously coexpressed in cells. This was specific, since SARS-CoV M did not retain an unrelated glycoprotein in the Golgi. Importantly, we found that an essential tyrosine residue in the SARS-CoV M cytoplasmic tail, Y(195), was important for S-M interaction. When Y(195) was mutated to alanine, M(Y195A) no longer retained S intracellularly at the Golgi. Unlike wild-type M, M(Y195A) did not reduce the amount of SARS-CoV S carbohydrate processing or surface levels when the two proteins were coexpressed. Mutating Y(195) also disrupted SARS-CoV S-M interaction in vitro. These results suggest that Y(195) is necessary for efficient SARS-CoV S-M interaction and, thus, has a significant involvement in assembly of infectious virus.
Asunto(s)
Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/fisiología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/fisiología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/fisiología , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Línea Celular , Proteínas M de Coronavirus , ADN Viral/genética , Aparato de Golgi/metabolismo , Aparato de Golgi/virología , Células HeLa , Humanos , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos/genética , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , Homología de Secuencia de Aminoácido , Glicoproteína de la Espiga del Coronavirus , Transfección , Tirosina/química , Proteínas del Envoltorio Viral/genética , Proteínas de la Matriz Viral/genética , Ensamble de Virus/genética , Ensamble de Virus/fisiologíaRESUMEN
Recent studies have highlighted the importance of the secretory pathway in stress-induced apoptotic signaling. Sensing stress at the endoplasmic reticulum and Golgi might first trigger recovery mechanisms, followed by apoptosis if repair is unsuccessful. Cleavage of endoplasmic-reticulum- or Golgi-resident proteins can signal repair or apoptosis and promote organelle disassembly during apoptosis. Initiation of apoptosis from the secretory pathway requires components of the death machinery localized to these membranes. Extensive trafficking between compartments of the secretory pathway might allow the cell to integrate signals and to determine the proper response to a particular stress.
Asunto(s)
Apoptosis/fisiología , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Transducción de Señal/fisiología , Estrés Fisiológico/metabolismo , Animales , Humanos , Membranas Intracelulares/metabolismo , Transporte de Proteínas/fisiologíaRESUMEN
Mgm1p is a conserved dynamin-related GTPase required for fusion, morphology, inheritance, and the genome maintenance of mitochondria in Saccharomyces cerevisiae. Mgm1p undergoes unconventional processing to produce two functional isoforms by alternative topogenesis. Alternative topogenesis involves bifurcate sorting in the inner membrane and intramembrane proteolysis by the rhomboid protease Pcp1p. Here, we identify Ups1p, a novel mitochondrial protein required for the unique processing of Mgm1p and for normal mitochondrial shape. Our results demonstrate that Ups1p regulates the sorting of Mgm1p in the inner membrane. Consistent with its function, Ups1p is peripherally associated with the inner membrane in the intermembrane space. Moreover, the human homologue of Ups1p, PRELI, can fully replace Ups1p in yeast cells. Together, our findings provide a conserved mechanism for the alternative topogenesis of Mgm1p and control of mitochondrial morphology.